1. Technical Field
This invention is related generally to the field of molecular and cellular biology. More particularly, this invention is related to fluorescent proteins and to methods for the preparation and use thereof.
2. Prior Art
Aequorea green fluorescent proteins (GFPs) have 238 amino acid residues in a single polypeptide chain. The native molecule has been shown to regenerate its intrinsic fluorescence from the totally denatured state. GFPs display a strong visible absorbance and fluorescence is thought to be generated by the autocyclization and oxidation of the chromophore having a tripeptide Ser-Tyr-Gly sequence at positions 65 to 67. Mutations to GFPs have resulted in various shifts in absorbance and fluorescence. The usefulness of GFPs stems from the fact that fluorescence from GFP requires no additional co-factors; the fluorophore is self-assembling via a cyclization reaction of the peptide backbone.
The chromophore of GFP is formed by the cyclization of the tripeptide Ser65-Tyr66-Gly67. This chromophore is located inside of the β-barrel that is composed of 11 anti-parallel strands and a single central α-helix. There are short helices capping the ends of the β-barrel. The chromophore has extensive hydrogen bonding with protein frame and can be affected by water molecules under the different folding status. The chromophore in a tightly constructed β-barrel exhibits absorption peaks at 400 and 480 nm and an emission peak at 510 nm with a quantum yield of about 0.72 when excited at 470 nm. The chromophore in enhanced green fluorescent protein (EGFP), which is GFP with a mutation S65T, has an improved fluorescence intensity and thermo-sensitivity.
Yellow fluorescent protein (YFP: S65G, V68L, S73A, T203Y), cyanide fluorescent protein (CFP: Y66W, N146I, M152T, V163A, N212K), and blue green fluorescent protein (BFP: Y66H, Y145F) are variants of GFP that differ in emission spectra and emission. Further, additional GFP variants, such as Venus, also have been constructed to have accelerated maturation and brightness. Due to the overlapping emission spectra and excitation spectra of GFP variants, fluorescence resonance energy transfer (FRET) from one to the other variants can be observed when the variants are in close proximity.
As GFPs may be cloned and expressed in a range of vectors across a diverse range of cells and organisms, GFPs are versatile tools for monitoring and visualizing physiological processes, protein localization, and expression of genes. GFPs are bio-compatible, and when used as a tag do not alter the normal function or localization of a protein to which they are fused. Proteins, cells and organelles marked with GFPs can be visualized and monitored in living tissue without the need for fixation. As such, it is possible to use GFPs to monitor and quantify the dynamics of cellular processes non-invasively in real time.
Accordingly, there is a need for improved fluorescent proteins that may be used in both in vivo and in vitro systems. Such fluorescent proteins should be able to detect changes in microenvironments so as to be useful as probes of cellular events involving changes in such microenvironments. Further, such fluorescent proteins should comprise a relatively short amino acid sequence that is relatively shorter than the sequence of natural GFPs, so that they may have applications in studies necessitating small proteins. In addition, the fluorescence signal of such fluorescent proteins should be able to be enhanced upon interaction with other peptides, proteins, or fragments. Further, there is a need for methods to produce fluorescent proteins exhibiting more efficient chromophore maturation. It is to these needs among others that the present invention is directed.